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Title: Complete structure, genomic organization, and expression of channel catfish (Ictalurus punctatus, Rafinesque 1818) matrix metalloproteinase-9 gene

Author
item Yeh, Hung-Yueh
item Klesius, Phillip

Submitted to: Bioscience Biotechnology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/24/2007
Publication Date: 3/23/2008
Citation: Yeh, H., Klesius, P.H. 2008. Complete structure, genomic organization, and expression of channel catfish (Ictalurus punctatus, Rafinesque 1818) matrix metalloproteinase-9 gene. Bioscience Biotechnology and Biochemistry. 72(3):702-714.

Interpretive Summary: Channel catfish production is the most important aquacultural industry in the southeastern United States. Its annual output reaches more than 450 million dollars. In the course of studying Edwardsiella ictaluri pathogenesis, we sequenced and identified the channel catfish matrix metalloproteinase-9 (MMP-9). The complete sequence of the channel catfish MMP-9 cDNA gene consisted of 2562 nucleotides. The open reading frame potentially encoded a 686-amino-acid peptide with a calculated molecular mass (without glycosylation) of approximate 77.4 kDa. In addition, channel catfish MMP-9 included a signal peptide with a cleavage site at positions 20 and 21 (AWS-HP), and had potentially heavy O-glycosylation sites between positions 447 and 490. However, channel catfish MMP-9 did not have the tripeptide Arg-Gly-Asp motif. The degree of conservation of the channel catfish MMP-9 amino acid sequence to human and mouse counterparts was 55%, while to those of other fish species was 67 – 74%. At the genomic DNA level, the full-length channel catfish MMP-9 DNA comprised 5663 nucleotides which are much shorter than human and mouse MMP-9 DNA. However, the exon-intron structure of channel catfish MMP-9 followed the splice acceptor/donor consensus rule, and the sequence contained 13 exons, sizes ranging from 104 (exon 12) to 239 (exon 2) nucleotides. The channel catfish MMP-9 genomic DNA did not have the alternating C and A sequence at the 5’-end UTR. The channel catfish MMP-9 transcript was constitutively expressed in restrictive tissues. This result will provide fundamental information for further exploration of the role of MMP-9 in fish pathophysiology as well as for the production of monoclonal/polyclonal antibodies for channel catfish MMP-9.

Technical Abstract: In the course of studying pathogenesis of enteric septicemia of catfish, we noted that the channel catfish (CC) matrix metalloproteinase-9 (MMP-9) expressed sequence tag (EST) was up-regulated after early Edwardsiella ictaluri infection. In this study, the CC MMP-9 gene was cloned, sequenced and characterized at both the cDNA and genomic DNA levels. The complete sequence of the CC MMP-9 cDNA gene consisted of 2562 nucleotides. Analysis of the nucleotide sequence revealed one open reading frame and 5’- and 3’-end untranslated regions (UTR). The 5’-end UTR had 103 nucleotides, while the 3’-end UTR had 398 bases in length. The CC MMP-9 mRNA instability motifs (ATTTA) that regulate mRNA degradation were located within the 3’-end UTR. In addition, a polyadenylation signal sequence (AATAAA) was located 24 bp upstream from the polyadenylation tail. The open reading frame potentially encoded a 686-amino-acid peptide with a calculated molecular mass (without glycosylation) of approximate 77.4 kDa. In addition, CC MMP-9 included a signal peptide with a cleavage site at positions 20 and 21 (AWS-HP), and had potentially heavy O-glycosylation sites between positions 447 and 490. However, CC MMP-9 did not have the tripeptide Arg-Gly-Asp motif. The degree of conservation of the CC MMP-9 amino acid sequence to human and mouse counterparts was 55%, while to those of other fish species was 67 – 74%. At the genomic DNA level, the full-length CC MMP-9 DNA comprised 5663 nucleotides which are much shorter than human and mouse MMP-9 DNA. However, the exon-intron structure of CC MMP-9 followed the splice acceptor/donor consensus rule, and the sequence contained 13 exons, sizes ranging from 104 (exon 12) to 239 (exon 2) nucleotides. The CC MMP-9 genomic DNA did not have the alternating C and A sequence at the 5’-end UTR. The CC MMP-9 transcript was constitutively expressed in restrictive tissues. The fused recombinant CC MMP-9 protein was expressed in E. coli, and confirmed by matrix assisted laser desorption ionization – time of flight (MALDI-TOF) mass spectrometry. This result will provide fundamental information for further exploration of the role of MMP-9 in fish pathophysiology as well as for the production of monoclonal/polyclonal antibodies for CC MMP-9.